Lecture 5

1. CELL SIGNALING AND MOTILITY (BIOL 3373) Lecture 5

2. Cytoskeleton

3. The cytoskeleton is a network of fibers extending throughout cytoplasm: It is built on three types of protein filaments: - Intermediate filaments (mechanical strength) - Actinfilaments (cell shape, locomotion) - Microtubules (support intracellular organelles, transport, chromosome segregation)

4. The cellular cytoskeleton is needed for many many cellular functions, such as: • Cell motility. • Muscle contraction. • Subcellular localization and anchoring of factors • Signaling and even gene transcription!

5. A cytoskeleton is not static but extremely dynamic. • constant turnover. • constant changing. • Remember: the interior compartments of the cell is also in constant motion, and it is the cytoskeleton which provides the means to allow this. • - Mitosis & meiosis • - Organelle movements • - Cell movement

6. Proteins that make up the fibers are very similar in all living things from bacteria to humans • For example: • tubulin (all cells) • actin (eukaryote cells) • The take home message: the cytoskeleton is ancient and essential for life

7. Aberrations of cytoskeleton organization are associated with human diseases Cytoskeleton is involved in essential cellular functions, therefore aberrations that modify cytoskeleton structure and organization are associated with a large variety of diseases including different types of cancers and neurodegenerative diseases. Immunolabeling of cytoskeleton elements (e.g. immunofluorescent staining) enables assessment of the cytoskeleton structure and represents a potent diagnostic tools Immunofluorescent staining of neural filaments (green) in fetal rat amygdala neurons Nuclei stained blue with DAPI. Double staining of Intermediate filaments vinculin (red) and microtubuli (green) in cultured chicken fibroblasts from Sigma- Aldrich

8. Aberrations of cytoskeleton organization are associated with human diseases Aβ amyloid plaques deposition, an hallmark of Alzheimer disease (AD), destroys cytoskeleton structure in neuronal axons impairing cell-cell communication. This condition is responsible for loss of memory in AD human neuronal-like cells in Alzheimer-induced condition human neuronal-like cells Immunofluorescent staining of neural filaments (green) in human neuronal-like cells in normal and Alzheimer-induced condition. Nuclei stained blue with DAPI. from Cimini et al 2012; D’Angelo et al 2010

9. Intermediate filaments

10. Intermediate Filaments • Found in the cytoplasm of most animal cells surrounding the nucleus and extending out into the cytoplasm. • Found extensively at cellular adhesion regions referred to as tight junctions (we will discuss it later!) • Found within the nucleus too - as part of the nuclear lamina which supports and strengthens the nuclear envelope

11. Intermediate Filaments • Have the great tensile strength. • Enable cells to withstand the forces and mechanical stress associated with stretching. • Called intermediate because their diameter is 10nm. • They are the toughest and most durable of the three types.

12. Consequence of losing intermediate fibers:

13. Intermediate Filaments

14. Intermediate filament structure • Strands are made of elongated fibrous proteins (A): • Each has a N-terminal globular head. • Each has a C-terminal globular tail. • Each has a central elongated rod domain, which is an extended alpha-helical region.

15. Intermediate filament structure • B)Two monomers around each other in a coiled-coil configuration forming a dimer.

16. Intermediate filament structure • C)two dimers further associated non-covalently with other dimers to form tetramers.

17. D)The tetramers associate together end-to-end and side-by-side by non-covalent interactions. Intermediate Filaments

18. Intermediate filament structure EEight tetramers associate together into a ropelike filament, making the final structure of the intermediate filament

19. Double click below https://www.youtube.com/watch?v=ll5MSxxHSCQ

21. Nuclear envelope Dynamic structure: The nuclear membrane come and goes as the cell undergoes cell cycle or division.

22. Nuclear envelope The nuclear envelope is supported by a meshwork of intermediate filaments which are formed from lamins.

23. Nuclear envelope • Note: • the assembly and disassembly of the lamina is • controlled by phosphorylation and dephosphorylation, respectively, of the lamins by protein kinases, i.e. during mitosis. • Tightly linked to signaling pathways!

24. Actin

25. Actin • The actin cytoskeleton is dynamic and reorganizes in response to intracellular and extracellular signals. • The polymerization of actin can provide forces that drive the: • extension of cellular processes • movement of some organelles

26. Actin • Found in all eukaryotic cells. • Actin represents approximately 5% of all the protein found in a cell. This is about 10-20% of the soluble protein fraction of a cell. • 100’s of proteins regulate organization of actin. • Bound to many membrane proteins.

27. Actin Actin filaments form many different cellular structures. Stress fibers microvilli Contractile ring lamellipodia

28. Actin • Also able to bind with a number of alternative actin-binding proteins to allow it to serve a variety of functions. • Proteins associated with the actin cytoskeleton produce forces required for cell motility. • Cell motility ( we will talk later!) is a fundamental and essential process for all eukaryotic cells.

29. Actin is a ubiquitously expressed cytoskeletal protein Actin is a ubiquitous and essential protein found in all eukaryotic cells. • Actin exists as: • a monomer called G-actin • a filamentous polymer called F-actin ( or actin Microfilaments)

30. G-Actin has subdomains 1-4. it binds to ATP, along with Mg++, within a deep cleft between subdomains 2 & 4.

31. Actin can hydrolyze its bound ATP  ADP + Pi, releasing Pi. The actin monomer can exchange bound ADP for ATP. The conformation of actin is different, depending on whether ATP or ADP is in the nucleotide-binding site.

32. G-Actin • Is a 43 Kd protein. Different isoforms of Actin: 2 isoforms in all non-muscle cells: – Beta (β) – Gamma (γ) 4 muscle isoforms in different muscle cells – Alpha (α) skeletal – Alpha (α) cardiac – Alpha (α) smooth – Gamma (γ) smooth

33. G- Actin The actin protein is highly conserved in mammals. Different ratios of β and γ exist in different cell types. Actin is a 374 amino acid protein. There is a four amino acid difference between the b and g isoforms at the N- terminal. Actin is a highly expressed gene.

34. G-actin (globular actin), with bound ATP, can polymerize to form F-actin (filamentous). F-actin may hydrolyze bound ATP  ADP + Pi & release Pi. ADP release from the filament does not occur because the cleft opening is blocked. ADP/ATP exchange: G-actincan release ADP & bind ATP, which is usually present at higher concentration than ADP in the cytosol.

35. F- Actin (MICROFILAMENTS) They are thin and flexible - about 7 nm in diameter. Each is a twisted chain of identical globular actin molecules - all pointing in the same direction - so they have what is termed a plus and minus end.

36. Actin filaments have polarity. The actin monomers all orient with their clefttoward the same end of the filament, called the minus end. The diagram above is oversimplified. Actin monomers spiral around the axis of the filament, with a structure resembling a double helix.

37. The polarity of actin filaments may be visualized by decoration with globular heads (S1) cleaved off of myosin by proteases. Bound myosin heads cause an appearance of arrowheads in electron micrographs.

38. In one experiment, short actin filaments were decorated with myosin heads. After removal of excess unbound myosin, the concentration of G-actin was increased, to promote further actin polymerization. Filament growth at one end, designated plus (+), exceeded growth at the other end, designated minus (-). In electron micrographs, bound myosin heads appear as arrowheadspointing toward the negative endof the filament. Barbed ends orient toward the plus end.

39. F- Actin (MICROFILAMENTS)

40. F- ACTIN FORMATION : NUCLEATION • De novo actin polymerization is a multistep process that includes a lag phase (nucleation),an elongation step and a steady phase. • Nucleation: two g actin monomers bind very weakly and they can dissociate from each others very easily. however addition of further actin monomers forms a stable oligomers (dissociation is unlike), therefore it acts as nucleusfor polymerization. • The nucleus assembly is very slow process, which explains the lag phase. • The nucleus once assembled accelerates the polymerization process

41. F- ACTIN FORMATION: ELONGATION AND STEADY PHASE • During the elongation the rates of monomer incorporation at the two ends is higher than monomer dissociation, resulting in the filament elongation. • the rates of monomer incorporation at the two ends are not equal: the barbed end of an actin filament is the fast growing end, therefore it determines the elongation of the actin filament in one direction. • In the steady state the growth of the polymer due to actin monomers addition balances the shrinkage of the polymer due to the disassembly back to the monomers.

42. Monomeric G actin addition and release Monomeric G actin binds to ATP Upon polymerization, actin ATPase activity cleaves ATP to ADP ATP hydrolysis acts as a molecular “clock” Older actin filaments have more G actin bound to ADP, therefore Filaments are unstable and disassemble Polymer disassembling: the shrinkage of the polymer due to the release of monomers is higher than growing due to monomer addition.

43. F- ACTIN FORMATION • Filament formation is dependent upon the level of ATP which binds to the G-actin subunits and on various ions such as magnesium. • Increased levels of Mg2+ favor the formation of filaments while lowered levels favor depolymerization. • G-actin subunits bound to ATP bind at the ends of the microfilaments with about 5X more binding at the (+) end as compared to the (-) end.

44. F- ACTIN FORMATION Note: Critical concentration is the concentration in which the rate of subunit addition equals the rate of subunit loos

45. Actin treadmilling Actin filaments may undergo treadmilling, in which filament length remains approximately constant (Steady phase), while actin monomers add at the (+) end and dissociate from the (-) end.

46. Toolsto study Actin Various agents produced by fungi and other organisms (and, some now by pharmaceutical companies) affect cells by altering the state of the cytoskeleton. Some of these specifically affect actin. Cytochalasins Phalloidin

47. Cytochalasin • Cytochalasins are fungal antibiotics. • They bind to filament ends thereby preventing addition of G-actin subunits. • The end result is the filaments disappear due to de-polymerization with no polymerization.

48. Phalloidin - Phalloidin is from Amanita phalloides. - They bind to filaments & prevents depolymerization resulting in a stabilization of the microfilaments in whatever situation they existed in at the time of drug addition. - Effectively used as a cell biological reagent to localize microfilaments (e.g., FITC-Phalloidin)

49. Toolsto study Actin These drugs are used to inhibit or stabilize actin filaments to understand their role in biological processes. For example, addition of cytochalasins causes the contractile ring of dividing cells to disappear thus inhibiting cytokinesis (cell division).

50. Effects of inhibitors of Actin Filaments